Microbiology Manual

LAB. MANUAL 14

MANUAL OF METHODS

OF

ANALYSIS OF FOODS

FOOD SAFETY AND STANDARDS AUTHORITY OF INDIA

MINISTRY OF HEALTH AND FAMILY WELFARE

GOVERNMENT OF INDIA

NEW DELHI

2012

MICROBIOLOGICAL TESTING

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MICROBIOLOGY OF FOODS 2012

MANUAL ON METHOD OF MICROBIOLOGICAL TESTING

TABLE OF CONTENTS S.No. Title Page No.

Chapter 1: Microbiological Methods 1. Aerobic Mesophilic Plate count 2. Aciduric Flat Sour Spore-formers 3. Bacillus cereus 4. Detection and Determination of Anaerobic Mesophilic Spore

Formers in Foods (Clostridium perfringens )

5. Detection and Determination of Coliforms, Faecal coliforms and E.coli in Foods and Beverages.

6. Direct Microscopic Count for Sauces, Tomato Puree and Pastes 7. Fermentation Test (Incubation test). 8. Rope Producing Spores in Foods 9. Detection and Confirmation of Salmonella species in Foods 10. Detection and Confirmation of Shigella species in Foods 11. Detection, Determination and Confirmation of Staphylococcus aureus

in Foods

12. Detection and Confirmation of Sulfide Spoilage Sporeformers in Processed Foods

13. Detection and Determination of Thermophilic Flat Sour Spore formers in Foods

14. Detection and Confirmation of Pathogenic Vibrios in Foods Estimation of Yeasts and Moulds in Foods Detectioin and Confirmation of Listeria monocytogenes in Foods

15. Bacteriological Examination of water for Coliforms Bacteriological Examination of water for Detection, Determination and Confirmation of Escherichia coli Bacteriological Examination of water for Salmonella and Shigella Bacteriological Examination of water for Clostridium perfringens Bacteriological Examination of water for Bacillus cereus Bacteriological Examination of water for Pseudomonas aeruginosa

16. Chapter 2: Culture Medias 17. Chapter 3: Equipment, Materials and Glasswares 18. Chapter 4: Biochemical Tests

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Microbiological Methods for Analysis of Foods, Water, Beverages and Adjuncts

Chapter 1

1. Aerobic Mesophilic Plate count

Indicates microbial counts for quality assessment of foods

1.2 Equipment:

Refer to Chapter 3 (Equipment, Materials & Glassware).

1.3 Medium:

o Plate count agar; o Peptone water 0.1%, o (Chapter 2 for composition of medium)

1.4 Procedure:

1.4.1 Preparation of food homogenate

Make a 1:10 dilution of the well mixed sample, by aseptically

transferring sample to the desired volume of diluent.

Measure non-viscous liquid samples (i.e., viscosity not greater than

milk) volumetrically and mix thoroughly with the appropriate volume of

diluent (11 ml into 99 ml, or 10 ml into 90 ml or 50ml into 450 ml).

Weigh viscous liquid sample and mix thoroughly with the appropriate

volume of diluent (11 + 0.1g into 99ml; 10+ 0.1g into 90ml or 50+0.1g into

450ml).

Weigh 50+0.1g of solid or semi-solid sample into a sterile blender jar

or into a stomacher bag. Add 450 ml of diluent. Blend for 2 minutes at low

speed (approximately 8000 rpm) or mix in the stomacher for 30-60 seconds.

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Powdered samples may be weighed and directly mixed with the diluent.

Shake vigorously (50 times through 30 cm arc).

In most of the food samples particulate matter floats in the dilution

water. In such cases allow the particles to settle for two to three minutes and

then draw the diluent from that portion of dilution where food particles are

minimum and proceed.

1.4.2 Dilution:

If the count is expected to be more than 2.5 x103 per ml or g, prepare

decimal dilutions as follows. Shake each dilution 25 times in 30 cm arc. For

each dilution use fresh sterile pipette. Alternately use auto pipette. Pipette

1 ml of food homogenate into a tube containing 9 ml of the diluent. From the

first dilution transfer 1ml to second dilution tube containing 9ml of the

diluent.

Repeat using a third, fourth or more tubes until the desired dilution is

obtained.

1.4.3 Pour plating:

Label all petriplates with the sample number, dilution, date and any

other desired information. Pipette 1ml of the food homogenate and of such

dilutions which have been selected for plating into a petri dish in duplicate.

Pour into each petri dish 10 to 12ml of the molten PCA (cooled to 42-45oC)

within 15 min from the time of preparation of original dilution. Mix the

media and dilutions by swirling gently clockwise, anti-clockwise, to and fro

thrice and taking care that the contents do not touch the lid. Allow to set.

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1.5 Incubation:

Incubate the prepared dishes, inverted at 35oC for 48+2 hours. (Or the

desired temperature as per food regulation e.g. in case of packaged drinking

water).

1.6 Counting Colonies:

Following incubation count all colonies on dishes containing 30-300

colonies and record the results per dilution counted.

1.7 Calculation

In dishes which contain 30-300 colonies count the actual number in

both plates of a dilution and as per the formula given below:

C N= (N1+0.1N2) D

C is the sum of colonies counted on all the dishes retained

N1 is the no. of dishes retained in the first dilution

N2 is the no of dishes retained in the second dilution

D is the dilution factor corresponding to first dilution

E.g.

At the first dilution retained (10-2):165 & 218 colonies

At the second dilution retained (10-3) 15 & 24

N= 165+218+15+24 = 422 = 19182 [2 + (0.1x2) x 10x-2] 0.022

Rounding the result to first two digits gives 19000 CFU.

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1.8 Expression of Result

Aerobic (Mesophilic) Plate Count = 19000 CFU/g or 1.9x104 CFU/g

or

If plates from all dilutions have no colonies and inhibitory substances have

not been detected, the result is expressed as less than 1 x 101 CFU per g or

ml.

If plates from the lowest dilutions contain less than 30 colonies, record the

actual number and calculate as above but express results as CFU per g or ml.

Note:- This method, as all other methods, has some limitations. Microbial

cells often occur as clumps, clusters, chains or pairs in foods, and may not

be well distributed irrespective of the mixing and dilution of the sample.

Moreover the single agar medium used, the conditions of incubation,

aeration etc., are not conducive to the growth of various populations of

bacteria that may be present in a food sample.

For statistical reasons alone, in 95% of cases the confidence limits of this

test vary from 12% to 37%. In practice even greater variation may be

found specially among results obtained by different microbiologists.

(Corvell and Morsettle, J. Sci. Fd. Agric., 1969, vol. 20 p 573)

References:

1. Official Methods of Analysis of AOAC International (1995). 16th

Edition. Edited by Patricia Cuniff. Published by AOAC International.

Virginia. USA. Test 17.2.01 p.3-4.

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2. Compendium of Methods for the Microbiological Examination of

Foods. (1992) Carl Vanderzant and Don F. Splittstoesser Eds.

Washington D.C. p. 75-87

3. Bacteriological Analytical Manual (1992) 6th Edn. Arlington, V.A.

Association of Official Analytical Chemists for FDA, Washington,

D.C. p. 17-21.

4. Microbiology- General guidance for the enumeration of

Microorganisms-Colony count technique at 35oC (first revision)

IS5402-2002, ISO4833:1991. Bureau of Indian Standards, Manak

Bhavan, 9 Bhadur Shah Zafar Marg, New Delhi110002.

2. To Determine and Confirm Aciduric Flat Sour Spore Formers in

Foods.

The organism of this group is Bacillus coagulans. It is responsible for

spoilage of canned products.

2.1Equipment:

Refer to Chapter 3 (Equipment, Material and Glassware)

2.2 Culture Media:

Dextrose tryptone agar (with bromocresol purple)

2.3 Procedure:

Weighed samples or dilutions of the sample are taken in a test tube

and heat shocked at 88oC for 5 min. The sample tubes are immediately

cooled and one ml of the heat shocked sample or decimal volume is

transferred to petri plates. 18 to 20 ml of melted bromocresol purple agar is

added. After mixing the plates are incubated at 55oC for 48h.

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Surface colonies on dextrose tryptone agar will appear slightly moist,

usually slightly convex and pale yellow. Subsurface colonies on this medium

are compact with fluffy edges. Colonies are surrounded by a yellow zone.

Suspected colonies are counted and expressed as number per g of the

sample.

2.4 Calculation

Average plate count x dilution factor = X


Aciduric flat sour spore formers = X/g

References:



Virginia. USA. Test. 17.6.03., p.44.



Washington.D.C.p.291-295.

3. Detection and Determination of Bacillus cereus in Foods, and

Beverages.

3.1 Equipment:

Refer to Chapter 3

3.2 Culture media and reagents

o Mannitol-egg yolk-polymyxin (MYP) agar o Trypticase-soy-polymyxin broth o Phenol red dextrose broth

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o Nitrate broth o Nutrient agar slants and plates o Nutrient agar with L-tyrosine o Nutrient broth with lysozyme o Modified Voges- Proskauer medium (VP) o Motility medium o Nitrate test reagents o Voges- Proskauer test reagents

3.3 Procedure

3.4 Preparation of food homogenate

Prepare as directed in 1.4.1 3.4.1 Dilution

Prepare decimal dilutions by pouring 1ml in 9 ml of dilution water. 3.4.A Most Probable Number Method

This procedure is suitable for the examination of foods which are expected

to contain fewer than 1000 B.cereus per g.

i. Inoculate each of three tubes of trypticase-soy-polymyxin broth with

1 ml food homogenate and its dilutions.

ii. Incubate at 30oC for 48 hours.

iii. Examine for dense growth typical of B.cereus

iv. Vortex-mix and using a 3mm loop transfer one loopful from each

growth positive tube to dried MYP medium plates. Streak to obtain

isolated colonies.

v. Incubate at 30oC for 48 hours

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vi. Pick one or more eosin pink (mannitol fermentation positive) colonies

surrounded by precipitate zone (due to lecithinase activity) from each

plate and transfer to nutrient agar slants for confirmation tests.

vii. The confirmed B.cereus count is determined using the MPN Table 4

of Test No. 10 for coliform count. On the basis of the number of tubes

at each dilution in which B.cereus was detected and reported as MPN

of B.cereus per gram.

3.4.B Plate Count Techniques This procedure is suitable for the examination of foods expected to

contain more than 1000 B. cereus per gram.

Inoculate duplicate MYP agar plates with the homogenate and each

dilution of homogenate by spreading 0.1 ml evenly on to each plate in

duplicate with sterile bent glass streaking rods (hockey sticks). Incubate

plates 24 hours at 30oC.

3.4.B.I Counting Colonies

The number of eosin pink colonies surrounded by lecithinase zone are

counted. If reactions are not clear, incubate plates for added 24 hours before

counting. Plates must ideally have 15-150 colonies.

Five or more colonies of presumptive B.cereus are picked from plates

and transferred to nutrient agar slants for confirmation (3.5).

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3.5 Confirmation Techniques

Gram Stain

Incubate the streaked nutrient agar slant either from for confirmation

for 24 hours at 30oC. Make Gram stain and examine under microscope. B.

cereus will appear as large Gram positive bacilli in short to long chains;

spores are ellipsoidal, central to sub-terminal and do not swell sporangium.

Biochemical tests

Transfer 3 mm loopful of this culture to a tube containing 0.5ml

sterile diluent. Vortex mix. Inoculate (or streak) the suspended culture into

the following media and read the biochemical reaction.

Table 3: Biochemical tests

Media Incubation at 35oC Typical Reaction Phenol red dextrose broth

Incubate anaerobically for 24 hours

Acid produced (color changes from red to yellow).

Nitrate broth For 24 hours Reduces nitrates to nitrites

Modified VP Medium For 48 hours Positive Nutrient agar with tyrosine

For 48 hours Positive

Nutrient broth with lysozyme

For 24 hours Growth positive

3.6 Calculations:

As per 1.6

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3.7 Reporting:

After confirmation, the number of B.cereus colonies is multiplied by

the reciprocal of the dilution that the countable plate represents (It should be

noted that the dilution factor is 10-fold higher than the sample dilution since

only 0.1 ml was plated) and report as B.cereus/gram.

3.8 Expression of Results:

Bacillus cereus= Present/Absent

References:



Virginia. USA Test 17.8.01 p.52-54.



Washington. D.C. p.593 603.

3. Bacteriological Analytical Manual (1992) 6th Edn. Arlingon. V.A.

Published by Association of Official Analytical Chemists for FDA,

Washington.D.C.p.191-198.

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4. Detection and Determination of Anaerobic Mesophilic Spore formers

(Clostridium perfringens).

4.1 Equipment and media 4.2 Equipment: Refer to Chapter 3. (Equipment, Materials and Glassware)

4.3 Media: Tryptone sulfite cycloserine agar (TSC),

Cooked meat medium

4.4 Procedure

Inoculate 2 g of food sample into 15 to 20 ml of cooked meat medium

in duplicates. Incubate at 35o for 24 h.

Positive tubes showing turbidity and gas production are streaked on to

TSC agar plates. Overlay with TSC agar. Incubate plates upright,

anaerobically for 18 to 20 h at 35oC.

Count all colonies that are black in color surrounded by a zone of

precipitate.

4.5 Confirmation

Inoculate a portion of the selected black colony from TSC agar on to

motility nitrate agar and lactose gelatin agar by stabbing. Also inoculate a

tube of fluid thioglycollate medium. Incubate at 35o C for 24 h.

Observe microscopically the culture growing in thioglycollate media

for the presence of large gram-positive rods. The culture is non-motile and

growth therefore occurs only along the line of inoculum in mobility nitrate

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agar, and they are positive for reduction of nitrate to nitrite which is

indicated by the development of red or orange color of the medium. On

lactose gelatin medium, the culture shows positive reaction for fermentation

of lactose as indicated by gas bubbles and change in color of medium from

red to yellow. Gelatin is liquified by C.perfringens.

4.6 Calculation

NA


Clostridium perfringens = present/absent

Reference:



Virginia. USA. Test No. 17.7.02 p. 48 50.


Foods. (1992) Carl Vanderzant and Don F. Splittstoesser. Eds.

Washington D.C. p. 623 635.

3. Bacteriological Analytical Manual (1992) 6th Edn. Arlington.V.A.

Association of Official Analytical Chemists for FDA,

WashingtonDC.p.209214.

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5. Detection, Determination and Confirmation of Coliforms, Faecal

coliforms and Escherichia coli in Foods and Beverages.

5.1 Equipment:

Refer to Chapter 3. (Equipment, Media and Glassware)

5.2 Culture media and reagents

Refer to test 5

5.3 Procedure

5.3.1 Test for Coliforms

Coliforms in foods may be enumerated by the solid medium method

or by the Most Probable Number (MPN) method.

5.3.1.A Solid medium method

Preparation of food homogenate

Prepare as directed under 1.4.1

5.3.1.A.1 Dilutions


5.3.1.A.2 Pour Plating

Pipette 1ml of the food homogenate (prepared sample) and of each

dilution into each of the appropriately marked duplicate petri dishes.

Pour into each petri-dish 10-12 ml of VRBA (tempered to 48oC) and

swirl plates to mix. Allow to solidify. Overlay with 3 to 5 ml VRBA and

allow to solidify.

Incubate the dishes, inverted at 35oC for 18 to 24 hours.

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5.3.1.A.3 Counting the colonies

Following incubation, count all colonies that are purple red in colour,

0.5 mm in diameter or larger and are surrounded by a zone of precipitated

bile acids. Optimally the plates should have 30 to 100 colonies.

5.3.1.A.4 Calculation

Multiply the total number of colonies per plate with the reciprocal of

the dilution used and report as coliforms per g or ml.

5.3.1.B Most Probable Number method

This method is valuable in those samples where coliform density is

low because higher quantity of sample can be used for examination. It is

based on probability statistics wherein aliquots of decimal volumes/dilutions

of the sample are transferred to several (1 to 5) tubes of specific medium.

Positive tubes are scored and the MPN estimate is directly made using the

Table 5.

5.3.1.B.1 Preparation of food homogenate

Prepare as directed under 1.4.1 5.3.B.1.2 Dilutions:


5. 3.B.1.3 Inoculation

Inoculate each of 3 tubes of LST broth (containing inverted Durham

tubes) with 1ml of food homogenate (1:10).

Carry out the same operation from the first (1 in 100) and the second

(1 in 1000) dilution tubes. Using a fresh sterile pipette for each dilution.

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5. 3.B.1.4 Incubation

Incubate the LST tubes at 35+0.5oC for 24 and 48 hours.

5.4 Presumptive test for coliforms

Record tubes showing gas production after 24 hours and reincubate

negative tubes for further 24 hours. Then record tubes showing gas

production.

5.5 Confirmed test for coliforms

Transfer a loopful from each gas positive tube of LST to a separate

tube of BGLB broth.

Incubate the BGLB broth tubes at 35+0.5o C for 48+2h.

The formation of gas confirms the presence of coliform bacteria.

Record the number of positive tubes that were confirmed as positive for

coliform.

5.6 Calculation

Note the MPN appropriate to the number of positive tubes from the

table 5.4.

For example:

3 in 1:10; 1 in 1:100 and 0 in 1:1000. The table shows that MPN=43

coliforms per g or ml.

Coliforms= present/absent per g

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Table 5. Most Probable Number (MPN) per 1 g of sample, using 3 tubes with each of 0.1, 0.01, and 0.001 g portions.

Positive tubes Positive Tubes Positive tubes Positive tubes

0.1 0.01 0.001 MPN 0.1 0.01 0.001 MPN 0.1 0.01 0.001 MPN 0.1 0.01 0.001 MPN 0 0 0 110

0

5.7.C Test for Faecal Coliforms

Proceed as directed from 5.5

Transfer a loopful from each gas positive tube of LST to a separate

tube of EC broth.

Incubate the EC tubes at 45.5+0.2oC in water bath for 24+2 hours.

Submerge broth tubes so that water level is above highest level of medium.

Record tubes showing gas production. 5.7.C.1 Calculations:

As directed under the test for coliforms.

5.8.D Test for Escherichia coli

(i) Proceed as directed under test for faecal coliforms.

Streak one plate L-EMB from each positive BGLB tube in a way to

obtain discrete colonies.

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Incubate inverted plates at 35 +0.5o C for 24+2 hours.

Examine plates for typical nucleated dark centered colonies with or

without sheen. If typical colonies are present pick two from each EMB

plate by touching needle to the center of the colony and transfer to a

PCA slant.

Incubate slants at 35+0.5o C for 18 to 24 hours

Transfer growth from PCA slants to the following broth for biochemical

tests (vide Chapter 4 under Biochemical Tests).

Tryptone broth: Incubate 24+2 hours at 35+0.5o C and test for indole.

MR-VP Medium: Incubate 48+2 hours at 35+0.5o C. Aseptically transfer

1ml of culture to a 13x100 mm tube and perform the Voges Proskauer test.

Incubate the remainder of MR-VP culture an additional 48h and test for

methyl red reaction.

Koser citrate broth: Incubate 96 hours at 35+0.5o C and record as + or

for growth.

LST broth: Incubate 48+2 hours at 35+0.5o C and examine for gas

formation.

Gram stain: Perform the Gram stain in a smear prepared from 18 hours

PCA slant. Presence of small red coloured rods confirms Escherichia coli.

Compute MPN of E.coli per g or ml considering gram negative,

nonspore forming rods producing gas in lactose and classify biochemical

types as follows (IMViC)(Table 5.1).

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Table 5.1: Micro-organism & IMViC

Indole MR VP Citrate Type + + - - Typical E. coli. - + - - Atypical E. coli. + + - + Typical intermediate - + - + Atypical Intermediate - - + + Typical Enterobacter aerogenes + - + + Atypical Enterobacter

aerogenes

5.8.D.1 Calculations

As per MPN table

5.8.D.2 Interpretation

Escherichia coli= x MPN/g

Reference:



Virginia. USA. Test No. 17.2.02, p. 4-5.



Washington D.C. p.325-341.

3. Bacteriological Analytical Manual (1992) 6th Edn. Arlington. V.A.


WashingtonD.C.p.2731.

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6. Direct Microscopic Count in Tomato Puree, Sauce, Paste, Chutney.

1. Howard Mold Count

2. Bacterial Count

3. Yeast and Bacterial Spore Count

6.1.1 Equipment: Refer to Chapter 3.

6.1.2 Special Equipment

i) Howard mould counting slide

(ii) Haemocytometer

The Howard mould counting slide is a thick glass slide with a flat

plane of rectangle of 20x15 mm in the middle of the slide, surrounded by a

moat flanked on each side by shoulders 0.1mm higher than flat plane

surface. The cover glass when placed is supported on the shoulders and

leaves a depth of 0.1mm between underside of cover glass and plane surface.

In the case of Haemocytometer the flat plane surface is ruled in the form of a

square with sides measuring 1mm each. This square is divided into 25

medium size squares and 400 small size squares.

6.2 Procedure:

6.2.A Mould Count Preparation of sample

Tomato juice: Use juice as it comes from container

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Catsup (Ketchup) or sauce: Place 50ml stabilizer solution in 100 ml

graduated cylinder, add 50ml well mixed sample by displacement and mix

thoroughly.

Stabilizer solution: 0.5% Sodium Carboxy Methyl Cellulose (NaCMC)

place 500ml boiling water in high speed blender. With blender running add

2.5gms NaCMC and 10ml formalin and blend for 1 minute. Keep in a

stoppered bottle (handle the blender carefully because hot materials in the

blender create pressure on closure with blender lid).

Puree and Paste: Dilute the sample with stabilizer solution and mix

thoroughly so that the refractive index of 1.3448 to 1.3454 at 20oC (or

1.3442 to 1.3448 at 25oC) is obtained.

6.2.A.1 Preparation of slide

Clean Howard slide so that Newtons rings are produced between

slide and cover glass. Remove cover and with knife, blade or scalpel, place

portion of well mixed sample upon central disk. Spread evenly over disk and

cover with cover glass to give uniform distribution. Discard any mount

showing uneven distribution or absence of Newtons ring or spillage of

liquid into moat.

6.2.A.2 Mould count

Place slide under microscope and examine with such adjustments that each

field of view covers 1.5 sq.mm obtained by so adjusting draw-tube that

diameter of field becomes 1.382 mm2. When such adjustment is not possible

make use of accessory drop in ocular diaphragm with aperture accurately cut

to necessary size. Diameter of area of field of view can be determined by use

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of stage micrometer. When instrument is properly adjusted, volume of liquid

examined per field is 0.15 mm3. Use magnification of 90-125X. Use

approximately 200X magnification to confirm identity of mould filament.

Prepare two mounts and count only 25 fields from each, observing in

such a manner as to be representative of all sections of mount. Observe each

field noting presence or absence of mould filament and recording results as

positive when aggregate length of not less than 3 filaments present exceeds

1/6 of diameter of field. In case a single filament is traversing several fields

of microscope it is counted as one positive field. For calculations refer 6.3.

6.2.B Bacterial count

Preparation of sample for bacterial count in the case of a

homogeneous food sample such as catsup and sauce involves proper

dilutions to facilitate easy identification and counting. Normally a 1:5

dilution would serve the purpose. To 20ml of distilled water in a 25 ml

graduated cylinder add 5 ml of sample by displacement and shake

thoroughly.

Place the haemcytometer slide under microscope and using 400 to 500

magnification count four small size squares from each corner of ruled

chamber and the central medium square (total 20 small squares). For

calculations refer 6.3.

6.2.C Yeast and Bacterial spore count

The same slide prepared for bacterial counts is used and a total of 200

squares comprising of 80, 40 and 80 from the top, middle and base of ruled

chamber respectively is counted. For calculations refer 6.3.

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6.2 Calculations

Calculation 6.2.A for Mould Count

Calculate proportions of positive fields from results of examination of

all observed field and report as percent fields containing mould filaments.

No. of positive fields Percent positive fields = -------------------------------------- X 100 No. of fields observed

Calculation 6.2.B for Bacterial count

No. of bacteria in 20 small squares = B

No. of bacteria in 400 small square= (B x 400) / 20

That is - 20 B bacteria

1.0 mm3 contains - 20 x 10B

1.0 cc contains - 20 x 10 x 103 B or 2 x 105 B

If the material is diluted five times then the number of bacteria per ml

of sample is

=5 x 2 x 105B or 10 x 105B or 106B or B million/cc.

e.g. If B = 20 then the count will be 20 million per cc.

Calculation 6.2.C for yeast and spores

Calculate number of yeasts/bacterial spores per 1/60 mm3 as follows:

No. of yeasts/spores in 200 small squares = Y

No. of yeast/spores in 400 small squares = (400 x Y)/200 or = 2Y

Or 0.1 mm3 contains = 2Y yeast

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1.0 mm3 contains = 2 x 10 Y yeasts

1/60 mm3 = (2 x 10 x 1 Y yeast)/60

Or 1/3 Y yeasts

If diluted 5 times then 5 x 1/3Y; or

5/3 Y yeasts/bacterial spores per 1/60 mm3 of the sample.

6.4 Expression of Results

Mould Hyphae positive fields = %

Microscopic Bacterial Count = 106 per cc

Yeast and Bacterial spores = 1/60 mm3

Reference:



Virginia. USA.



Washington D.C. p. 97-104.

7. Fermentation Test (Incubation test). To determine commercial sterility of processed canned foods.

7.1 Equipment:

Refer to Chapter 3. (Equipment, Materials and Glassware)

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7.2 Media:

o Tryptone broth o Cooked meat medium o Orange serum broth o Potato dextrose agar o APT broth

7.3 Procedure

The most reliable test for determining commercial sterility of a

container of a product is to incubate that container in an appropriate

temperature, long enough to allow any significant microorganisms contained

therein to grow and to manifest their presence. This is the incubation or

fermentation test.

7.4 Routine Production Monitoring

For low acid products destined for storage at temperatures above

40oC, containers from each sampling period or retort load should be

incubated at 55oC for 5 to 7 days.

For all other low-acid products incubate at 30oC to 35oC for 10 days.

For acid or acidified foods incubate at 25o to 30oC for 10 days.

7.5 Examination

Containers may be removed from the incubator whenever outward

manifestations of microbial growth appear (e.g., swells or with transparent

containers, noticeable product change). At the end of the incubation period,

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some containers should be opened to detect possible flat sour spoilage by

measure of reduced pH as compared to good packs.

Weigh each suspect container to the nearest gram. Subtract the

average tare weight of the empty container and determine net weight.

Before opening, the container must be cleaned with detergent and

water, rinsed and wiped dry with clean paper towels.

Containers are opened employing aseptic techniques with extra

precautions. Note abnormal odour, consistency changes, and frothiness.

Measure pH electrometrically or colorimetrically.

7.6 Sub culturing of Product Samples

Transfer about 2g of product from each container to media mentioned

below. Tubes for anaerobes should be exhausted in flowing steam for an

exposure of 20 min and cooled to 55oC prior to inoculation if not freshly

prepared and autoclaved. For detection of molds in high acid foods, potato

dextrose agar pour plates are prepared. Measure pH of the product and

observe product odor and appearance.

7.6.A Low acid foods (pH > 4.5 or 4.5)

Medium Incubation temperature and time Organism

Tryptone broth 30 to 35oC for 5 days Mesophilic aerobes Tryptone broth 55oC for 5 days Thermophilic

aerobes Cooked meat medium 30 to 35oC for 5 days Mesophilic

anaerobes Cooked meat medium 55oC for 5 days Thermophilic

anaerobes

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7.6.B High acid Foods (pH 4.5 or


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8. Rope Producing Spores in Flours.

8.1 Requirements of the procedure

8.2 Equipment and media

8.3 Equipment:


8.4 Culture Media:

Dextrose tryptone agar

8.5 Procedure:

Fifty grams of the sample is weighed and transferred to 450 ml of sterile

0.1% peptone water in a blender jar for mixing. Alternately a stomacher may

also be used for mixing.

Ten and one ml volumes of the peptone water suspension are pipetted

into separate 100ml portions of melted dextrose tryptone agar contained in

250 ml flasks and held at 45oC. The flasks and a control flask is submerged

in a boiling water bath for 15 mins.

After heating, the flask contents are cooled to about 45oC and contents

of each flask is poured into 5 sterile plates in approximately equal volumes.

When the agar has solidified, the plates are inverted and incubated at 35oC

for 48h.

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8.6 Calculation:

Count as rope producing organisms, the surface colonies that are grey-

white, vesicle like, becoming at first drier and finally wrinkled. Add to this

count any subsurface colony that displays stringiness when tested. The total

colonies on the set of 5 plates from the flask that received 10ml suspension

are considered as rope spores per gm of sample.


Rope spores = /g

References:

1. Compendium of Methods for the Microbiological Examination of Foods.

(1992) Carl Vanderzant and Don F. Splittstoesser.

Eds.WashingtonD.C.p.265-274.

9. Detection and Confirmation of Salmonella species in foods.

9.1 Equipment: Refer to Chapter 3. (Equipment, Materials and Glassware)

9.2 Culture Media:

o Lactose broth o Trypticase Soy Broth o Trypticase Soy Broth Containing Potassium Sulfite at a final

concentration of 0.5%.

o Reconstituted Non-Fat Dry Milk o 1% aqueous Brilliant Green Dye Solution. o Selenite Cystine Broth

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o Tetrathionate Broth o Xylose Lysine Deoxycholate (XLD) Agar o Hektoen Enteric Agar (HEA) o Bismuth Sulphite Agar (BSA) o Triple Sugar Iron (TSI) Agar o Lysine Iron Agar (LIA) o Urea Broth o Phenol Red Dulcitol Broth o Phenol Red Lactose Broth o Tryptone Broth o KCN Broth o Malonate Broth o Buffered Glucose (MR-VP) Medium o Brain Heart Infusion (BHI) Broth o Buffered Peptone Water

9.3 Procedure:

9.4 Preparation of sample and pre-enrichment

Aseptically open the sample container and weigh 25g sample into a

sterile empty wide mouth container with screw cap or suitable closure.

Add 225ml of sterile lactose broth to the sample. Buffered peptone

water, Trypticase soy broth, and nutrient broth can also be used for pre-

enrichment. Make a uniform suspension by blending if necessary. Cap

container and let stand at room temperature for 60 min. Instead of lactose

broth the recommended pre-enrichment broth for the following food samples

is as follows :

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Non fat dry milk and dry whole milk Sterile distilled water. Add

0.45 ml of 1% aqueous briliant green dye before incubation.

Dried active yeast Trypticase soy broth

Onion-garlic powder Trypticase soy broth containing potassium sulfite at a

final concentration of 0.5%

Milk Chocolate Reconstituted non fat dry milk.

Shake and adjust pH (if necessary) to 6.80.2 with sterile 1N NaOH or 1N

HCl.

Incubate at 35oC for 242 hours

9.5 Selective enrichment

Gently shake incubated sample mixture and transfer 1 ml to 10 ml of

selenite cystine broth and an additional 1 ml to tetrathionate broth. Incubate

242 hours at 35oC.

9.6 Selective media plating

Vortex mix and streak 3 mm loopful of incubated selenite cystine

broth on selective media plates of XLD, HEA and BSA. Repeat with 3mm

loopful of incubated tetrathionate broth.

Incubate plates at 35oC for 242 hours and 482 hours.

Observe plates for typical Salmonella colonies

On XLD (after 24h) - Pink colonies with or without black centres.

On HEA (after 24h) - Blue green to blue colonies with or without black

centers.

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On BSA (after 24 to 48h) Brown, grey or black colonies sometimes

with metallic sheen. Surrounding medium is usually brown at first, turning

black with increasing incubation time.

9.7 Treatment of typical or suspicious colonies

Pick with needle typical or suspicious colonies (if present) from each

XLD, HEA and BSA plates. Inoculate portion of each colony first into a TSI

agar slant, streaking slant and stabbing butt and then do the same into an

LIA slant.

Incubate TSI and LIA slants at 35oC for 24+2 hours and 48+2h

respectively. Cap tubes loosely to prevent excessive H2S production.

Table 9.7.A Typical Salmonella reactions are :

TSI LIA

Slant Alkaline (red) Alkaline (Purple)

Butt Acid (Yellow) Alkaline (Purple)

H2S production (blackening in butt) + or - +

A culture is treated as presumptive postive if the reactions are typical

on either or both TSI and LIA slants.

9.8 Biochemical tests

Using sterile needle inoculate a portion of the presumptive positive

culture on TSI slant into the following broths. Incubate at 35oC for the

specified period of days and read for Salmonella typical reactions.

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Table 9A: Biochemical tests

Broth/ Media Time of incubation

Results

Urea broth 24+2h Negative (no change in yellow colour of medium)

Phenol red lactose broth

48+2h *Negative for gas and/or acid reaction

Phenol red sucrose broth

48+2h *Negative for gas and/or acid reaction

Phenol red dulcitol broth

48+2h *Postive for gas and/or acid reaction

Tryptone broth 24+2h Negative for indole test KCN broth 48+2h Negative (no turbidity) Malonate broth 48+2h *Negative (green colour unchanged) MR-VP medium 48+2h Negative for VP test but positive for MR test. *(Note : Majority of S. arizonae are atypical for these reactions).

Table 9B: Criteria for discarding Non-Salmonella Cutlures

Test(s) or Substrate(s) Results Urease test Postive (purple-red) Indole test Positive (red) Flagellar test (Polyvalent or spicer-Edwards

Negative (no agglutination)

Lysine decarboxylase test Negative (yellow) KCN broth Positive (growth) Phenol red lactose broth* Positive (acid and/or gas)** Lysine decarboxylase test Negative (yellow) Phenol red sucrose broth Positive (acid and/or gas)** Lysine decarboxylase test Negative (yellow) KCN broth Positive (growth) Voges-Proskauer test Positive (red) Methyl red test Negative (yellow)

* Malonate broth positive cultures are tested further to determine if they are

Salmonella arizonae

** Do not discard positive broth cultures if corresponding LI agar cultures

give typical Salmonella reactions; test further to determine if they are

Salmonella sp. (vide 9).

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9.9 Serological Tests

To reduce number of presumptive positive cultures (TSI positive and

urease negative) carried through biochemical identification tests, the

following serological flagellar (H) screening test may be carried out.

Transfer 3mm loopful of culture into 5ml of BHI broth and incubate at

35oC until visible growth occurs (About 4-6 hours).

Add about 2.5ml formalized physiological saline solution.

Test with Salmonella flagellar (H) antisera. Positive cultures show

visible agglutination.

Further confirmation can be made by using Salmonella Polyvalent (O)

antiserum.

9.10 Calculation:

NA

9.11 Expression of Result:

Salmonella = Present/Absent per 25 g

References:



Virginia. USA. Test. 17.9.01 p. 55 62.



Washington D.C. p.371-422.



D.C.p.5169.

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10 Detection and Confirmation of Shigella species

10.1 Equipment:


10.2 Culture media:

o Gram Negative (GN) Broth o MacConkey agar o Xylose Lysine Deoxycholate (XLD) Agar o Triple Sugar Iron (TSI) Agar slants o Urea Broth o Acetate Agar Slants o Carbohydrate Fermentation Media o Tryptone Broth (for Indole test) o Buffered Glucose (MR-VP) Medium o Kosers Citrate Broth o Decarboxylase Test Media with Lysine or Ornithine o Motility Test Medium o Thornleys Semi-Solid Arginine Medium.

10.3 Procedure:

10.4 Enrichment:

Using aseptic techniques mix or blend if necessary 25 g sample with

225 ml of gram negative broth. Transfer to a sterile 500 ml bottle.

Adjust pH (if necessary) to 6.0 - 7.0 with sterile 1N NaOH or 1N HCl.

Incubate at 35-37oC for 18 hours.

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10.5 Selective streaking:

Transfer a 5mm loopful of the enrichment broth culture to the surface

of MacConkey agar and XLD agar plates and streak to obtain isolated

colonies.

Invert and incubate plates at 35-37oC for 24+2h. Typical Shigella

colonies on XLD agar appear as red or pink colonies usually about 1mm in

diameter and on Mac Conkey agar as opaque or transparent colonies.

Inoculate each suspected colony into TSI agar slant by streaking the

slant and stabbing the butt. After overnight incubation at 35-37oC, typical

Shigella reaction is alkaline (red) slant and acid (yellow) butt with no H2S or

gas production:

10.6 Other Biochemical tests to confirm Shigella

Perform the following biochemical tests on a portion of the suspected

culture on the TSI slant noted in 9.7 & 9.8

Table 10

Test Reaction Urease - Motility - Acetate utilization - Gas from glucose - IMVIC Reaction + + - - or - + - - Lysine decarboxylase - Arginine dihydroloase - or + Ornithine decarboxylase + or -


Shigella = Present / Absent per 25g of sample

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References:





Association of Official Analytical Chemists for FDA, Washington

D.C. p. 71 76.

11. Detection, Determination and Confirmation of Staphylococcus

aureus.

11.1 Equipment:

Refer to Chapter 3. (Equipment, Materials and Glassware).

11.2 Culture media:

o Tripticase (tryptic) soy broth with 10% sodium chloride and 1% sodium pyruvate.

o Baird Parker (BP) Medium o Brain Heart Infusion (BHI) Broth o Desiccated Coagulase Plasma (rabbit) with EDTA o Butterfields Buffered Phosphate Diluent o Plate Count Agar (PCA)

11.3 Procedure:

11.3.1 Preparation of food homogenate:

Aseptically weigh 50 g food sample into the sterile blender jar. Add 450ml

of diluent (1:10) and homogenize 2 min at high speed (16000-18000 rpm).

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Alternately use stomacher for sample preparation 11.3.2 Dilution:

Pipette 10ml of the food homogenate into 90ml of diluent (or 1ml to

9ml) to make a 1:100 dilution. Mix well using a vortex-mixer.

Transfer 1ml from this dilution to a fresh tube of 9ml to give a 1:1000

dilution. Repeat until the desired dilution is obtained.

11.3.A Most probable number method:

This procedure is recommended for testing processed foods likely to

contain a small number of S. aureus.

11.3.A.I Inoculation:

Inoculate each of 3 tubes of tryptose soy broth (with 10% sodium

chloride and 1% sodium pyruvate) with 1ml of food homogenate.

Carry out the same operation from the first and subsequent dilutions

using a fresh sterile pipette each time.

Maximum dilution of sample must be high enough to yield negative

end point. Incubate at 35oC for 48h.

11.3.A.II Surface Streaking:

Vortex mix the tubes from .A.1 and then using 3mm loop transfer one

loopful from each growth positive tube to dried BP medium plates. Streak so

as to obtain isolated colonies. Incubate at 35-37oC for 48 hours.

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11.3.A.III Interpretation:

Colonies of S. aureus are typically grey black to jet black, circular,

smooth, convex, moist and 2-3 mm diameter on uncrowded plates.

Frequently there is a light colored (off-white) margin, surrounded by opaque

zone (precipitate) and frequently with outer opaque zone (precipitate) and

frequently with outer clear zone; colonies have buttery to gummy

consistency when touched with the inoculating needle.

11.3.A.IV Confirmation techniques:

Using a sterile needle, transfer (noting the dilution) at least one

suspected colony from each plate to tubes containing 5ml BHI and to PCA

slants.

Incubate BHI cultures and slants at 35oC for 18-24h.

Perform coagulase test on the BHI cultures. Retain slant cultures for

repeat tests.

11.3.A.V Reporting:

Coagulase positive cultures are considered to be S. aureus. Now

record number of positive tubes (and the respective dilutions) of S. aureus.

Report most probable number (MPN) of S. aureus per gram from Table 4 of

MPN values.

11.3.B Surface Plating method :

This method is applicable for general purpose use in testing foods

expected to contain > 10 cells of S. aureus per g.

Transfer 1ml of the food homogenate (1:10 dilution) and other

dilutions to triplicate plates of BP medium and equitably distribute 1ml

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inoculum over the triplicate plates. Spread inoculum over agar surface using

sterile bent glass streaking rods (hockey sticks).

Incubate plates in upright position in the 35-37oC incubator for about

1 hour or until inoculum is absorbed by medium. Then invert plates and

incubate 45-48 hours.

11.3.B.1 Counting colonies:

Count colonies of typical S. aureus appearance (as described in

19.5.3.3). Test for coagulase production on suspected colonies. Add number

of colonies on triplicate plates represented by colonies giving positive

coagulate test. Multiply the count obtained by inverse of corresponding

sample dilution. Report as S. aureus per gm or ml of the sample.

11.3.B.2 Expression of result:

Staphylococcus aureus = x/g

References:



Virginia. USA Test. 17.5.01 p.32 34.






D.C. p. 161 165.

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12. Detection and Confirmation of Sulfide Spoilage Spore formers in

Processed Foods.

12.1 Equipment:


12.2 Cuture Media:

Sulfite agar

12.3 Procedure :

For sample preparation and heat treatment follow the steps mentioned for

anaerobic thermophilic spore formers. (Test No. 4)

Inoculations of the prepared sample are placed into the sulfite agar

medium with a nail. Incubate at 55oC for 24 to 48h in anaerobic jar.

D. nigrificans will appear as jet-black spherical areas, the color due to

the formation of iron sulfide. No gas is produced. Colonies can be counted

and reported as spores/g of sample.

12.4 Calculation:

Number of colonies x dilution factor


Spores of sulfide spoilage/g

References:

Compendium of Methods for the Microbiological Examination of Foods.

(1992) Carl Vanderzant and Don F. Splittstoesser. Eds.

WashingtonD.C.p.317-323.

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13. Detection and Determination of Thermophilic Flat Sour

Sporeformers.

13.1 Equipment:


13.2 Culture Media:

Dextrose Tryptone Agar.

13.3 Procedure:

Weighed samples or dilution are heat treated at 100oC for 30 min

followed by rapid cooling. Aliquots of these solutions are then transferred to

petri plates. Dextrose tryptone agar is added and swirled gently to distribute

the inoculum. Allowed to solidify. The inverted plates are incubated at 55oC

for 48 to 72 hr.

In case of starch samples the dilutions are added directly to sterile

dextrose tryptone agar (100 ml) contained in flasks. The flasks are

autoclaved at 5lb for 10min. The flasks are then gently agitated while

cooling as rapidly as possible. The entire mixture is distributed equally into

5 plates and allowed to harden. It is then layered with a thin layer of sterile

plain 2% agar in water and allowed to harden. The inverted plates are

incubated at 50 oC to 55oC for 48 to 72 hr.

Flat sour colonies are round, are 2 to 5mm in diameter, show a dark,

opaque center and usually are surrounded by a yellow halo in a field of

purple.

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The colonies are counted and expressed in terms of number of spores

per g of the sample.

13.4 Calculation:

=Number of colonies x dilution factor


Thermophilic flat sour bacteria = x/g

References:



Virginia. USA



Washington D.C. p. 299 307

14. Detection and Determination of Pathogenic Vibrios in Foods.

14.1 Equipment:


14.2 Culture Medium:

o Thiosulphate Citrate Bile Salts Sucrose Agar o Gelatin Phosphate Salt Broth and Agar.

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o Kligler Iron Agar. o T1 N1 Agar.

14.3 Procedure:

14.4 Enrichment

Weigh 25g sample and transfer to 225ml of GPS broth. Incubate at

35oC for 6 to 8 h.

14.5 Plating:

Prepare dried plates of TCBS and GPS agar medium. Transfer a

loopful of the surface growth of the broth culture to the surface of the two

plating medium and streak in a manner that will yield isolated colonies.

Incubate plating medium for 18 to 24 h at 35oC.

14.6 Interpretation:

Typical colonies of V.cholerae on TCBS agar are large (2 to 3 mm in

diameter) smooth, yellow (occasional slow sucrose fermentors are green),

and slightly flattened with opaque centers and translucent peripheries. On

GPS agar the colonies have a cloudy zone around them that becomes more

definite after a few minutes of refrigeration. In oblique light, the colonies

appear iridescent green to bronze colored and finely granular.

Typical colonies of V. parahaemolyticus on TCBS agar appear round,

opaque, green or bluish colonies, 2 to 3 mm in diameter.

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14.7 Confirmation:

Subculture all suspect colonies of V. cholerae on to T1N1 agar and

incubate at 35oC for 24h. Stab streak a KIA slant with the culture and

incubate the KIA slant overnight at 35oC. V. cholerae cultures have an

alkaline (red) slant and an acid (yellow) butt, no gas and no blackening in

the butt. Also perform the string test on suspect cultures as follows.

Emulsify a large inoculum from the T1 N1 agar culture in a large drop of

0.5% sodium desoxycholate in 0.85% saline solution. Within 60 seconds, a

mucoid mass forms and this material strings when a loopful is lifted (up to 2

to 3cm) from the slide. Further confirmation is by serological reactions.

Stab streak suspect colonies of Vibrio on the TSI slant and incubate

overnight at 35oC. Typical reaction of V. parahaemolyticus is an alkaline

slant and an acid butt but no gas or H2S production

14.8 Results:

Test for pathogenic Vibrios = Positive/ Negative

Reference:



Virginia. USA, Test No. 17.11.01 p. 108 110.






D.C.p.111121.

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15. Estimation of Yeasts and Molds in Foods and Beverages.

15.1 Equipment:


15.2 Media:

o Potato Dextrose Agar o Mycophilic Agar o Antibiotic Solution

15.3 Procedure:

Prepare food homogenate and decimal dilutions as directed under

1.4.1 and 1.4.2 respectively.

15.4 Pour plating:

Label all petri plates with the sample number, dilution, date and any

other described information.

Pipette 1ml of the food homogenate of such dilutions which have been

selected for plating into a petri dish in duplicate.

Acidify PDA or malt agar with sterile 10% tartaric acid to pH 3.5+0.1.

Do not reheat medium once acid has been added. Pour 10-12 ml of the agar

medium (tempered to 45oC). Mix by swirling and allow to solidify.

(OR)

Add 2ml antibiotic solution to 100ml of plate count, mycophil or malt

agar. Mix and pour 10-12ml of the agar medium tempered to 45oC. Mix by

swirling and allow to solidify.

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15.5 Incubation:

Invert plates and incubate at 20 or 25oC for 2 to 5 to 7 days. Discard

plates after seven days of if growth is not observed, observe plates every day

and mark the colonies because some time fungal growth spreads to entire

plate and mask the colonies. Do not open the plates which are showing

fungal sporangia.

15.6 Counting colonies:

Count colonies, multiply by the inverse of the corresponding dilution

and report as yeast or mould count per g or ml.

15.7 Reporting:

Yeast and Mould count = x/g

Reference:






D.C. p.227-230.

16. Detection and confirmation of Listeria monocytogenes in Food

Warning: while testing L. monocytogenes it is recommended that a properly

equipped laboratory under supervision of skilled Microbiologist is done. The

material used during testing is carefully disposed off after sterilization.

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Pregnant personnel may be asked to avoid handling of L. moncytogens

cultures and undertaking the tests.

16.1 Equiupments:

Refer chapter 3

16.2 Culture media and reagents:

o Phosphate buffered peptone water o Half Frazer broth o Frazer broth o Modified Oxford Agar o PALCAM Agar o Tryptone Soya Yeast Extract Agar o Tryptone Soya Yeast Extract Broth o Sheep Blood Agar o Carbohydrate utilization broth (Rhamnose and Xylose) o Motility Agar o CAMP Medium and test organisms o Hydrogen peroxide solution

16.3 Preparation of test sample:

Take 25 g of a well mixed sample in stomacher bag and use 225 ml of

Half Frazer broth. Stomach the sample for two minutes. Pour aseptically the

contents in to a wide mouth bottle and incubate at 30C for 242h (a black

coloration may develop).

Take one ml of the above culture and transfer to 9ml of Frazer broth.

Incubate the inoculated tube at 37C for 48 2h at 35-37C.

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From 24 h culture of Half Frazer broth and 48h Frazer broth streak out

culture on Modified Oxford Agar and PALCAM agar so that well separated

colonies are obtained.

Invert the plates and incubate at 35 or 37C for 24 h and if required an

additional 18 h if growth is slight or no colonies appear. Examine the plates

for colonies presumed to be L. monocytogenes.

16.4 Appearance of colonies:

On M Ox agar the colonies are small (1mm) greyish surrounded by a

black halo.

After 48 h the colonies turn darker with a possible green luster and are

about 2 mm in diameter with black halos and sunken centres.

On PALCAM agar after 24 h the colonies appear1.5 to 2 mm in

diameter greyish green or olive green some times with black centre and

always surrounded by a black halo and depressed centre.

16.5 Confirmation of Listeria species:

Select five typical colonies from one plate of each medium. If

presumed colonies are less than five on a plate, take all of them.

Streak the selected colonies from each plate on to the surface of a well

dried TSYEA for obtaining well separated colonies. Invert the plates and

incubate at 35C or 37C for 18 to 24 h or until the growth is satisfactory.

Typical colonies are 1 mm to 2 mm in diameter, convex, colorless and

opaque with an entire edge. Carry out the following tests from colonies of a

pure culture on the TSYEA.

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Catalase reaction:

With the help of loop pick up an isolated colony and place it in H2O2

solution on a glass slide. Immediate production of gas bubbles indicates

catalase positive reaction.

Gram staining:

Perform Gram staining on a colony Listeria are Gram positive slim

short rods.

Motility Test:

Take colony from TSYEA plate and suspend it TSYE broth. Incubate

at 25C for 8 to 24 h until cloudy medium is observed. Take a drop of

culture and place it on a glass slide. Cover the top with a cover slip and

observe under a microscope. Listeria is seen as slim rods with a tumbling

motility (cultures grown above 25C fail to show this motion. Compare them

with a known culture cocci or large rods with rapid motility are not

Listeria.

As an alternative stab motility agar tube with an isolated colony from

TSYEA and incubate at 25C for 48 h. typical umbrella like appearance

around the stab indicate motility positive culture. If growth in not positive

incubate up to five days and observe for the stab again.

16.6 Confirmation of Listeria monocytogenes:

Heamolysis test:

Take a colony from TSYEA and stab it on a well dried surface of

sheep blood agar plate. Simultaneously stab positive (L. monocytogenes) and

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negative (L. innocua) control cultures. Inver the plates and incubate at 35C

or 37C for 242 h. examine the plates.

L. monocytogenes show clear light zones of beta haemolysis.

L. innocua does not show any haemolysis. Examine the plates in a bright

light to compare test cultures with the controls.

Carbohydrate utilization:

Inoculate each of the carbohydrate utilization broths (rhamnose and

xylose) with a culture from TSYE broth and incubate at 35 C or 37v for

upto 5 days. Appearance of yellow color indicates a positive reaction

within24 to 48 h.

CAMP test

On a well dried surface of sheep blood agar streak each of the

Staphylococcus aureus and Rhodococcus equi cultures in single lines and

parallel to each other and diametrically opposite, a thin even innoculum is

required.

Streak the test strain separated in a similar manner at right angles to

these cultures as that the test strain and S. aureus and R.equi cultures do not

touch but their closest are about 1 mm or 2 mm apart. Several test strains can

be streaked on the same plate. Simultaneously streak control cultures of L

monocytogenes, L innocua and L. ivanovii. Incubate plates at 35 to 37 oC for

18 to 24 h.

Observe plates against bright light. In L. monocytogenes case there is

enhanced zone of beta haemolysis at the intersectiono of S. aureus.

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L. innocua does not show any enhanced zone of haemolysis with S.

aureus or R. equi.

In case of L. ivanovii enhamced beta zone of haemolysis is seen on R.

equi side.

16.7 Interpretation of results:

All Listeria species are small, Gram positive rods that demonstrate

motility and catalase positive reaction. L monocytogenes are distinguished

from other species by the characteristics listed in table given below.

Table 16 Species Haemolysis Production of

acid with Rhamnose

Production of acid with

Xylose

CAMP Test S. aureus R equi

L.monocytogenes + + - + - L innocua - V - - - L. ivanovii + _ + - + L. seeligeri (+) - + (+) - L welshmeri - V + - - L grayi sub species grayi

- - - - -

L..grayi subspecies murrayi

- V - - -

16.8 Expression of results:

Based on the observations and interpretation of the results report

presence or absence of L. monocytogenes in test portion specifying the mass

in grams or mililitres of the sample taken.

L. monocytogenes =present or absent/ g or ml.

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17.Bacteriological Examination of Water for Coliforms

17.1 Equipment:

Refer to Chapter 3.

17.2 Culture Media:

Lauryl Sulphate Tryptose (LST) broth tubes of single and double

strength with inverted Durham tubes (10 ml quantities in tubes and 50 ml in

bottles).

17.3 Procedure: Dechlorination:

Samples collected in pre-sterilised bottles are mixed well. To every

100ml portion of chlorinated samples 0.1 ml of a 10% sterile solution of

sodium thiosulphate is added.

Various combinations of sample volume are taken depending on the

probable load of coliform in the sample. To 10 ml or 50 ml volume of

double strength broths, equal volumes of sample is added. To 10ml of single

strength broths 1 ml or 0.1 ml is added.

Tubes/bottles are incubated at 35oC for 24 and 48 hrs.

17.4 Interpretation

Record tubes showing gas production after 48 hrs. The MPN index

per 100ml sample is determined using the following statistical tables.

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Table 17A: MPN index and 95% Confidence limits for various combinations of positive and negative results when five 10 ML portions

are used.

No. of tubes giving positive reaction out of 5

of 10 ml each

MPN Index/100 ml 95% confidence Limits (Approximate)

Lower Upper 0 < 2.2 0 6.0 1 2.2 0.1 12.6 2 5.1 0.5 19.2 3 9.2 1.6 29.4 4 16.0 3.3 52.9 5 > 16.0 8.0 Infinite

Table 17B: MPN index and 95% Confidence limits for various combinations of positive and negative results when Ten 10 ML portions

are used

No. of tubes giving positive reaction out of 10

of 10 ml each

MPN Index/100 ml 95% Confidence Limits (Approximate)

Lower Upper

0 < 1.1 0 3.0 1 1.1 0.03 5.9 2 2.2 0.26 8.1 3 3.6 0.69 10.6 4 5.1 1.3 13.4 5 6.9 2.1 16.8 6 9.2 3.1 21.1 7 12.0 4.3 27.1 8 16.1 5.9 36.8 9 23.0 8.1 59.5 10 > 23.0 13.5 Infinite

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Table 17C: MPN Index and 95% Confidence Limits for Various combinations of positive results when five tubes are used per dilution

(10 ml, 1.0 ml, 0.1 ml) Combina-

tion of Positives

MPN Index/100 ml

95% Confidence Limits

(Approximate)

Combina-tion of

Positives

MPN Index/100 ml

95% Confidence Limits

(Approximate) Lower Upper Lower Upper

0-0-0 < 2 - - 4-2-0 22 9.0 56 0-0-1 2 1.0 10 4-2-1 26 12 65 0-1-0 2 1.0 10 4-3-0 27 12 67 0-2-0 4 1.0 13 4-3-1 33 15 77

4-4-0 34 16 80

1-0-0 2 1.0 11 5-0-0 23 9.0 86 1-0-1 4 1.0 15 5-0-1 30 10 110 1-1-0 4 1.0 15 5-0-2 40 20 140 1-1-1 6 2.0 18 5-1-0 30 10 120 1-2-0 6 2.0 18 5-1-1 50 20 150

5-1-2 60 30 180

2-0-0 4 1.0 17 5-2-0 50 20 170 2-0-1 7 2.0 20 5-2-1 70 30 210 2-1-0 7 2.0 21 5-2-2 90 40 250 2-1-1 9 3.0 24 5-3-0 80 30 250 2-2-0 9 3.0 25 5-3-1 110 40 300 2-3-0 12 5.0 29 5-3-2 140 60 360

3-0-0 8 3.0 24 5-3-3 170 80 410 3-0-1 11 4.0 29 5-4-0 130 50 390 3-1-0 11 4.0 29 5-4-1 170 70 480 3-1-1 14 6.0 35 5-4-2 220 100 580 3-2-0 14 6.0 35 5-4-3 280 120 690 3-2-1 17 7.0 40 5-4-4 350 160 820

4-0-0 13 5.0 38 5-5-0 240 100 940 4-0-1 17 7.0 45 5-5-1 300 100 1300 4-1-0 17 7.0 46 5-5-2 500 200 2000 4-1-1 21 9.0 55 5-5-3 900 300 3900 4-1-2 26 12 63 5-5-4 1600 600 5300

5-5-5 > 1600 --- ---

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17.5. Expression of Results:

Coliforms = x MPN/250 ml or 100ml

17.5.1 Plating Technique:

Alternately take desired volume of water (250 ml or 100 ml) and pass it

through a micropore filter of 0.2U. Take the filter disk and place on a well

dried surface of LST Agar. Incubate at 35C for 24 to 48 hrs. Count the

typical colonies and express the result as given below.

17.5.2 Expression of Result:

Coliform count = x cfu/g

References:

1. Standard Methods for the Examination of water and waste water.

(1989). 17th Edition. Edited by Lenore. S. Clesceru; Arnold. E.

Greenberg and R. Rhodes Trussell. Test 9221. P. 66 - 76



Washington D.C. p. 28 31

18. Bacteriological Examination of Water for Detection, Determination

and Confirmation of Escherichia coli.

18.1 Equipment:

Refer to Chapter 3.

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18.2 Culture Media:

Refer to test No. 5.

18.3 Procedure:

Take 250ml or 100 ml (as per the requirement of standard). Pour 50

ml of sample in 50 ml of double strength LST broth in five bottles

containing inverted tubes, or 20 ml of sample in 20 ml of double strength

LST broth in sugar tubes containing inverted Durham tubes. Incubate the

tubes at 35C for 24 to 48 hrs. Note the number of bottles/tubes showing gas

formation. Refer MPN tables from test No. 17 for calculation of number of

presumptive +ve E. coli .Proceed further as per test No. 5 for confirmation

of E. coli

18.4 Calculation:

As per test No. 17


Escherichia coli = present/ absent in 250ml or 100 ml.

18.6 Filteration Technique:

Alternately filter the required volume through a 0.2U micropore filter

and place it on VRBA Mc Conkey agar plate and incubate at 35C for 24 to

48 hrs. Count typical colonies and select five such colonies for confirmation

as per test No. 5.

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References:

1. Standard Methods for the Examination of water and wastewater.

(1989). 17th Edition. Edited by Lenore. S. Clesceru; Arnold. E. Green

berg and R. Rhodes Trussell. Test 9221. P. 66 - 76



Washington D.C. p.325 369.

19. Bacteriological examination of water for presence of Salmonella and

Shigella

19.1 Equipment:

Refer to Chapter 3.

19.2 Culture Media:

Refer to Test No. 9 and 10.

19.3 Procedure:

Refer to test no. 9 and 10.

Expression of Result:

Test for Salmonella = present or absent/250 ml

Test for Shigella = present or absent/ 250 ml

References:



Virginia. USA. Test 17.9.01 p. 55-62.

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Washington D.C. p.371 431 .



D.C. p. 51 69.

20. Bacteriological examination of water for Detection and

Confirmation of Clostridium perfringens

20.1 Equipment:



o Cooked Meat Medium o Tryptone sulfite Cycloserine Medium

20.3 Procedure:

Take 50 ml of sample water and pour in to five tubes of Cooked Meat

medium (de-chlorinate the sample if required. Put the tubes in water bath

maintained at 80 C for 15 minutes. Plug the tubes with vespar and incubate

at 35 C for 18 to 24 hrs. Formation of gas bubble below the wax seal

indicates anaerobic growth.

From each positive tube take a loopfull of culture and streak on to a

TSC medium. Overlay a thin layer of TSC agar. Incubate the inverted plates

at 35 C 18 to 24 hrs. Appearances of black colonies surrounded by a black

zone are Clostridium perfringens colonies.

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20.4 .Expression of Results:

Clostridium perfringens = present or absent/50 ml

References



Virginia. USA. Test. 17.7.02 p. 48 50.



Washington D.C.p. 623 635.



D.C. p. 209 214.

21. Bacteriological Examination of water - Bacillus cereus.

21.1 Equipment:



Refer to Test No. 6

21.3 Procedure:

Take 250 ml of water sample and pour 50 m in five bottles containing

50ml of double strength Trypticase Soy Polymixin broth. Incubate at 30 C

for 48 hrs and proceed as per test No.3.3.

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21.4. Calculations:

If desired estimate MPN as per table No. 17.1.


Bacillus cereus = present absent/250 ml

References:



Virginia. USA. Test 17.8.01 p. 52-54.






D.C.p191198.

22. Bacteriological analysis of water for Detection and determination of

Pseudomonas aeruginosa in water

22.1 Equipment:

Refer chapter 3

22.2 Culture media:

o Pseudomaonas presumptive test broth-single strength and Concentrated.

o Milk agar

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o Gelatin medium o Starch agar

22.3 Presumptive test:

Add 250 ml of water sample in to five bottles (50 ml in each)

containing 50 ml of concentrated Pseudomonas presumptive test broth.

Incubate the bottles at 371 C for 48 h. examine for growth and blue

green fluorescence under an ultra violet lamp in a darkened room or UV

light chamber.

22.4 Confirmation:

Take a loopful of culture above growth from each container and streak

onto a milk agar plate. Invert the plate and incubate at 420.5C for 24h.

Examine the plate for growth, pigment production and casein hydrolysis

(zone of clearance around colonies).

22.5 Enumeration:

All containers showing either growth or fluorescence which yield

typical colonies (after subculture on milk agar plates) shall be considered

positive for Pseudomonas aeruginosa.

Use table no. 4 for calculation of MPN count.

22.6 Non pigmented strains:

If growth is observed in the Pseudomonas presumptive test broth and

casein is digested in the Milk agar then inoculate a loopful from the broth

into gelatin medium and incubate at 371C.

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Oxidative reaction can be confirmed by Hough and Liefson test.

Nitrate is reduced to ammonia from the breakdown of acetamide.

From the presumptive test inoculate Milk agar and incubate the

inverted plate at 421C for 24h . Positive reaction for Pseudomonas

confirms atypical Pseudomonas aeruginosa in the sample.

Table 22: For reactions of Pseudomans aeruginosa. S.No. Mode of reaction

Typical Atypical Casein hydrolysis + + Growth at 42C + + Blue green fluorescence + - Gelatine liquefaction + + Nitrate reduction to ammonia + + Oxidative reaction in Hough and Liefson medium + +


Reference:

Packaged Natural Mineral Water Specifications (second revision) IS13428-

2005. Manak Bhavan, 8 Bahadur Shah Zafar Marg, New Delhi-110002.

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Culture Media

Chapter 2

Acetate agar Sodium chloride ................................................ 5.0 g Magnesium sulfate ........................................... 0.1 g Monoammonium phosphate ............................ 1.0 g Dipotassium phosphate .................................... 1.0 g Sodium acetate ................................................. 2.0 g Bromothymol blue ........................................... 0.08 g Agar ................................................................. 20.0 g Distilled water ................................................... 1.0 liter Mix ingredients in distilled water and heat gently to dissolve.

Dispense 7 ml portions into 16 x 150 mm tubes.

Sterilize at 121oC for 15 minutes, and slant the tubes to obtain a 1 inch

butt and a 1.5 inch slant, pH 6.8+0.2.

Baird-Parker medium

Basal medium Tryptone ........................................................... 10.0 g Beef extract ....................................................... 5.0 g Yeast extract ..................................................... 1.0 g Glycine ............................................................. 12.0 g Lithium chloride 6H2O ..................................... 5.0 g Agar ................................................................. 20.0 g

Suspend ingredients in 950 ml distilled water.

Boil to dissolve completely. Dispense 95.0 ml portions in screw

capped bottles. Autoclave 15 minutes at 121oC. Final pH 6.8-7.2 at 25oC.

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Bismuth Sulfite Agar

Peptone ............................................................. 10.0 g Beef extract ...................................................... 5.0 g Dextrose ........................................................... 5.0 g Disodium phosphate ......................................... 4.0 g Ferrous sulfate .................................................. 0.3 g Bismuth ammonium citrate ............................. 1.85 g Sodium sulfite ................................................... 6.15 g Agar ................................................................. 20.0 g Brilliant green ................................................... 0.025 g Distilled water ................................................... 1.0 liter

Dissolve ingredients in distilled water by boiling approximately 1

minute. Adjust to pH 7.7+0.2, cool to 45 to 50oC, suspending precipitate

with gentle agitation, and pour plates without sterilizing medium. Let plates

dry with covers partially open. Caution: Plates lose selectivity after 72 hours.

Brain Heart Infusion Broth Calf brain, infusion from ................................. 200.0 g Beef heart, infusion from ................................. 250.0 g Proteose peptone or polypeptone ..................... 10.0 g Dextrose ........................................................... 2.0 g Sodium chloride ............................................... 5.0 g Disodium phosphate ........................................ 2.5 g Distilled water .................................................. 1.0 liter Dissolve ingredients in distilled water by bringing to a boil. Dispense

into tubes and autoclave for 15 minutes at 121oC. Final reaction should be

pH 7.4+0.2.

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Brilliant Green Lactose Bile Broth 2% Peptone ............................................................. 10.0 g Lactose ............................................................. 10.0 g Oxbile ............................................................... 20.0 g Brilliant-green .................................................. 0.0133 g Distilled water .................................................. 1.0 liter

Dissolve the peptone and lactose in 500ml of distilled water, add the

ox bile dissolved in 200ml of water, mix and make up to 975 ml, and adjust

pH to 7.4+0.1. Add 13.3 ml of 0.1% aqueous solution of brilliant green. Add

distilled water to bring the total volume to 1 liter. Dispense in 10ml portions

into 20 x 50 mm test tubes containing inverted Durham tubes. Sterilize for

15 minutes at 121oC.

Bromocresol Purple Carbohydrate Broth Basal Medium Peptone ............................................................ 10.0 g Beef extract (optional) ..................................... 3.0 g Sodium chloride ............................................... 5.0 g Bromocresol purple .......................................... 0.04 g Distilled water .................................................. 1.0 liter

Dissolve the desired carbohydrate (5.0 g or 10.0 g glucose, 5.0 g

adonitol, 5.0 g arabinose, 5.0 g mannitol 5.0 g maltose, 5.0 g sucrose, 5.0 g

lactose, 5.0 g sorbitol, 5.0 g cellobiose, 5.0 g salicin, 5.0 g trehalose or

raffinose) per liter of basal medium. Adjust pH to 7.00.2. Dispense 8 ml

aliquots to 16 x 150 mm tubes containing inverted 12 x 75 mm tubes.

Autoclave 10 minutes at 121oC. Allow autoclave temperature to drop

slowly.

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Buffered Peptone Water Peptone ............................................................. 10.0 g Sodium chloride ............................................... 5.0 g Disodium hydrogen phosphate ........................ 9.0 g Potassium dihydrogen phosphate .................... 1.5 g Distilled water .................................................. 1.0 liter

Adjust pH to 7.0, dispense in portions of 225ml into bottles of 500ml

capacity and of 9ml in tubes. Sterilize for 20 min at 121oC.

Butterfields Buffered Phosphate Diluent Stock solution :

Monopotassium hydrogen phosphate .............. 34.0 g Distilled water .................................................. 500.0 ml

Adjust to pH 7.2 with about 175 ml sodium hydroxide solution dilute

to one liter. Sterilize at 121oC for 15 minutes and store in refrigerator.

Diluent

Dilute 1.25 ml stock solution to 1.0 liter with distilled water. Prepare

dilution blanks in suitable containers. Sterilize at 121oC for 15 minutes.

Cooked Meat Medium Beef heart .......................................................... 454.0 g Proteose peptone ............................................... 20.0 g NaCl .................................................................. 5.0 g Glucose ............................................................. 2.0 g

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Finely chop beef heart. Add approximately 1.5 g of heart particles to

test tubes. Add remaining components to distilled water and bring volume to

1.0 L. Mix thoroughly. Distribute into tubes in 10 ml volumes. Autoclave for

15 min at 121oC.

Czapek Yeast Autolysate (CYA) Agar Sucrose .............................................................. 30.0 g Agar .................................................................. 15.0g Yeast extract ..................................................... 5.0 g NaNO3 ............................................................... 5.0 g K2HPO4 ............................................................. 1.0 g KCL .................................................................. 0.5 g MgSO4.7H2O .................................................... 0.5 g FeSO4.7H2O ...................................................... 0.01 g pH 7.3+0.2 at 25oC

Add sucrose to 100ml distilled water and autoclave for 15 mins at

121oC. Cool to 50oC. Add the other components to 900 ml distilled water.

Autoclave for 15 mins at 121oC. Aseptically add the sterile sucrose solution

after it has cooled to 50oC.

Decarboxylase Test Media

Basal for use with Lysine, Arginine, Ornithine Moeller method (1954,

1955)

Basal medium

Peptone ............................................................. 5.0 litre Beef extract ...................................................... 5.0 g Bromocresol purple (1.6%) ............................. 0.625 ml Cresol red (0.2%) ............................................. 2.5 ml Glucose ............................................................ 0.5 g Pyridoxal .......................................................... 5.0 mg Distilled water .................................................. 1.0 liter

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The basal medium is divided into four equal portions, one of which is

tubed without the addition of any amino acids. These tubes of basal medium

are used for c

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Microbiology Manual